Peroxidase, because of its high turnover rate, good stability, and availability is widely used in enzyme-based analytical methods. For example, horseradish peroxidase (HRP) (EC 1.11.1.7) catalyzes the oxidation of a large variety of hydrogen-donating substrates with hydrogen peroxide. HRP is also one of the preferred enzymes for use in catalyzed reporter deposition.
Catalyzed reporter deposition is a novel method of signal amplification which constitutes the subject matter of U.S. Pat. Nos. 5,196,306 and 5,583,001. It is also discussed in Bobrow et al., Journal of Immunological Methods, 125: 279-285 (1989) and in Bobrow et al., Journal of Immunological Methods, 137: 103-112(1991).
The method utilizes an analyte-dependent enzyme activation ("ADEAS") to catalyze the deposition of additional reporter enzyme onto the solid phase, resulting in signal amplification and improved assay detection limits. In a preferred embodiment, HRP is the ADEAS.
The ADEAS reacts with a conjugate consisting of a detectably labeled substrate specific for theADEAS. When the ADEAS and the conjugate react, an activated conjugate is formed which deposits covalently wherever receptor for the activated conjugate is immobilized. The receptor is not reactive with the analyte-dependent enzyme activation system.
Conjugates can be synthesized using conventional coupling and labeling techniques. Substrate choice will depend upon the ADEAS selected. Thus, detailed knowledge is required of the catalytic properties of each specific enzyme in order to properly design a useful synthetic substrate and, if necessary, a receptor. Examples of conjugates which have been described include substituted phenols such as biotin tyramine, fluorescein tyramine, NADP, phosphorylated biotin, etc.
However, care must be exercised in designing substrates suitable for peroxidase-mediated assays. Guilbault et al., Analytical Chemistry, Vol. 40, No. 8, pages 1256-1263 (1968) studied a wide variety substrates for fluorometric determination of oxidative enzymes such as peroxidase, galactose oxidase, glucose oxidase, and invertase. These researchers reported that 3,4-dihydroxycinnamic acid, which contains a double bond in the side chain was not a useful substrate for the fluorometric determination of oxidative enzymes because it stopped the reaction with the enzyme.